Scanning Transmission Electron Microscopy for Imaging Extended Areas

ABSTRACT

A scanning transmission electron microscope for imaging a specimen includes an electron beam source to generate an electron beam. Beam optics are provided to converge the electron beam. A stage is provided to hold a specimen in the path of the electron beam. A beam scanner scans the electron beam across the specimen. A controller may define one or more scanning areas corresponding to locations of the specimen, and control one or more of the beam scanner and stage to selectively scan the electron beam in the scanning areas. A detector is provided to detect electrons transmitted through the specimen to generate an image. The controller may generate a sub-image for each of the scanning areas, and stitch together the sub-images for the scanning areas to generate a stitched-together image. The controller may also analyze the stitched-together image to determine information regarding the specimen.

CLAIM FOR PRIORITY

This application is a continuation of application Ser. No. 13/303,121,filed Nov. 22, 2011, which is incorporated herein by reference in itsentirety.

INCORPORATION OF SEQUENCE LISTING BY REFERENCE

This application incorporates by reference the contents of a 406 bytetext file created on Jan. 19, 2012, and named “Sequence.txt,” which isthe sequence listing for this application.

TECHNICAL FIELD

This application relates to scanning transmission electron microscopyfor imaging extended areas.

BACKGROUND

In certain applications it is desirable to use electron microscopy tosequence a polymer. Electron microscopy can theoretically be used, forexample, to sequence bases of a nucleic acid, such as the bases of astrand of deoxyribonucleic acid (DNA). The polymer is labeled at itsstructural units and stretched onto a substrate. An electron microscopeis then used to scan the polymer and thereby generate an image, whichcan be analyzed to identify the labels. Based on the correspondence ofthe label types with the structural unit types to which they bond, thepolymer can be sequenced.

Using conventional transmission electron microscopy (TEM) for sequencingmay, however, suffer from an undesirably low ratio of signal from thelabels to noise from the substrate or other causes. Images generated byelectron microscopy may also not be optimally focused at every portionof a polymer strand, which can detract from the ability to locate andidentify the labels. Moreover, performing conventional transmissionelectron microscopy may take an undesirably long time for polymersequencing. The throughput may be especially low for sequencing longpolymers, such as a full human genome, in any practical amount of time.

Thus, it is desirable to provide electron microscopy with asignal-to-noise ratio that is sufficiently good for polymer sequencing.It is also desirable for the electron microscope images to be focused atevery part of a polymer strand being examined. Moreover, it is desirablefor the electron microscopy to have a substantially high throughput tosequence the polymer sufficiently fast to be practical.

SUMMARY

In one embodiment, a scanning transmission electron microscope isprovided for imaging a specimen. The microscope comprises an electronbeam source to generate an electron beam. The microscope has beam opticsto converge the electron beam, a stage to hold a specimen in the path ofthe electron beam, and a beam scanner to scan the electron beam acrossthe specimen. The microscope also has a detector to detect electronstransmitted through the specimen to generate an image. A controller isprovided to (1) define one or more scanning areas corresponding tolocations of the specimen, (2) control one or more of the beam scannerand stage to selectively scan the electron beam in the scanning areas,(3) generate a sub-image for each of the scanning areas, (4) stitchtogether the sub-images for the scanning areas to generate astitched-together image, and (5) analyze the stitched-together image todetermine information regarding the specimen.

In another embodiment, a method is provided of imaging a specimen. Themethod includes generating an electron beam, converging the electronbeam, and holding a specimen on a stage in the path of the electronbeam. The method further comprises defining one or more scanning areascorresponding to locations of the specimen, and controlling one or moreof the beam scanner and the stage to selectively scan the electron beamin the scanning areas. Electrons transmitted through the specimen aredetected to generate a sub-image for each of the scanning areas. Themethod additionally comprises stitching together the sub-images for thescanning areas to generate a stitched-together image, and analyzing thestitched-together image to determine information regarding the specimen.

In yet another embodiment, a scanning transmission electron microscopeis provided for imaging a specimen. The microscope comprises an electronbeam source to generate an electron beam. The microscope has beam opticsto converge the electron beam into a longitudinally stretched beam, astage to hold a specimen in the path of the electron beam, and a beamscanner to scan the electron beam across the specimen. The microscopealso has a detector to detect electrons transmitted through the specimento generate an image. A controller is provided to (1) define one or morescanning areas corresponding to locations of the specimen, (2) controlone or more of the beam scanner and stage to selectively scan theelectron beam in the scanning areas, (3) generate a sub-image for eachof the scanning areas, (4) stitch together the sub-images for thescanning areas to generate a stitched-together image, and (5) analyzethe stitched-together image to determine information regarding thespecimen.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and aspectsof the transmission electron microscopes described herein and, togetherwith the description, serve to explain the principles of the invention.

FIG. 1 is a schematic diagram of an example of an embodiment of ascanning transmission electron microscope (STEM).

FIG. 2 is a three-dimensional schematic diagram of an example of anembodiment of condenser lenses containing multipole condenser elementsshaping an electron beam into a longitudinally stretched probe.

FIG. 3A is a plot of an example of the distribution of the electron beamin FIG. 2 across the A axis.

FIG. 3B is a plot of an example of the distribution of the electron beamin FIG. 2 across the B axis.

FIG. 4A is a schematic side view of an example of an embodiment oflenses that shape an electron beam into a longitudinally stretched probewith enhanced current.

FIG. 4B is another schematic side view of the embodiment illustrated inFIG. 4A, from a perspective orthogonal to the perspective of FIG. 4A.

FIG. 5 is a schematic top view of an example of an embodiment of asample that includes polymer strands on a substrate and a longitudinallystretched electron beam probe being scanned along the length of one ofthe polymer strands.

FIG. 6 is a side view of a schematic diagram of a portion of an exampleof an embodiment of a STEM in which a beam scanner electronically shiftsan electron beam from a first position to a second position.

FIG. 7 is a three-dimensional schematic perspective view of an exampleof an embodiment of a stage supporting a specimen that can be moved backand forth along at least two orthogonal axes in relation to themicroscope.

FIG. 8 is a schematic diagram of an example of an embodiment of a fieldof view that encloses a single defined scanning area covering a regionof a sample that includes multiple polymer strands.

FIG. 9 is a schematic diagram of a wider perspective on scanning area ofFIG. 8, showing a plurality of partially overlapping scanning areas.

FIG. 10 is a schematic diagram of another example of an embodiment of aplurality of fields of view, wherein multiple scanning areas are definedwithin each individual field of view.

FIG. 11 is a schematic diagram of yet another example of an embodimentof a field of view, wherein the field of view encloses a plurality ofscanning areas that are arranged as elongated strips overlying polymerstrands of a sample.

FIG. 12 illustrates an example of an embodiment of a mapping between theidentified presence of each of multiple DNA labels and theircorresponding nucleotide types, showing the sequence (SEQ ID NO:1) thatresults from the mapping.

DETAILED DESCRIPTION

A scanning transmission electron microscope (STEM) may be adapted andused advantageously to sequence polymers. The polymer may include anucleic acid, such as for example oligonucleotides and polynucleotides,DNA, or RNA of genomic, recombinant, or synthetic origin, which may besingle- or double-stranded, and represent sense or antisense strands, orany DNA-like or RNA-like material or other polymers, such as proteins,natural, recombinant, or synthetic in origin, and which may contain anynucleic acid, including variants such as 5-Metyl Cytosine and otherepigenetically modified bases, artificially modified bases, andindividual amino acids, both natural and artificial.

To facilitate STEM imaging of the polymer, structural units of strandsof the polymer may be labeled with atoms or molecules that facilitatedetection and identification. For example, the labels may be heavy atomsor clusters of atoms. Different labels may be associated with differenttypes of structural units of the polymer, respectively. For example,each type of label may be chosen to bond selectively to a correspondingtype of structural unit. Heavy atoms used for labeling may, in oneversion, have an atomic number of at least 55 to provide a desirablecontrast during imaging. Examples of atoms that can be used for labelinginclude Cl, Br, I, U, Os, Pb, Au, Ag, Fe, Pt, Eu, Pd, Co, Hg, Gd, Cd,Zn, Ac, W, Mo, Mn, Rb, Cs, Ra, Ba, and Sr.

After the polymer has been labeled, it is presented for imaging in amanner suitable for obtaining images of the different labels in relationto their positions along the polymer strands. For example, the polymerstrands may be stretched out on a substrate. This may involvestraightening the polymer. The polymer may also be attached to thesubstrate, such as by bonding. The STEM sequentially scans an electronbeam across the polymer, herein referred to as the sample. In this way,one or more images may be generated. These images may be evaluated, suchas by a controller of the STEM, to identify the labels in relation totheir location on the polymer. The identities of the labels can be usedto identify the structural units of the polymer, thus sequencing thepolymer.

A particularly useful application of the STEM is sequentially imaging aDNA strand in order to determine the sequence of the nucleotide basepairs of the DNA. For example, a single strand of DNA may be stretchedin preparation for imaging using techniques that have been described inPCT Publication No. WO 2009/046445 dated Sep. 4, 2009, entitled“Sequencing Nucleic Acid Polymers with Electron Microscopy,” and filedas International Application No. PCT/US2008/078986 on Jun. 10, 2008(this PCT publication is hereby incorporated by reference in itsentirety). A particular set of nucleotides may be labeled using a labelthat contains at least one heavy scatterer. Examples of such labelsinclude osmium, triosmium, and platinum.

In the case of a double-stranded nucleic acid, such as DNA, the labelsmay be attached to either one strand or both strands of the nucleicacid. If only one strand is to be labeled, the sequence of the otherstrand may be inferred from the sequence of the imaged strand. Acomplementary strand, namely a second strand with nucleotidescomplementary to a first strand, may be formed for the purpose oflabeling and imaging in lieu of the first strand. The two strands of thenucleic acid may be separated from each other by, for example,denaturing processes, such as thermal and enzymatic, that can be used tobreak the hydrogen bonding between the strands. Alternatively, a singlestrand may be synthesized from a template. For example, polymerase chainreaction (PCR) or reverse transcriptase processes may be utilized forthis purpose. In yet another version, a single strand may be chemicallysynthesized one nucleotide at a time, such as in an oligonucleotidesynthesis process. A single strand can also be obtained by purificationfrom a natural source, such as RNA from cells. If the strand issynthesized, the labels may be attached to the nucleotides beforesynthesis. The labels may be selected from types that do not obstructany step of the synthesis, such as the polymerase reactions.

If both strands are labeled and imaged, each base pair can be identifiedtwice using different labels. This redundancy can provide protectionagainst failure to label a nucleotide as well as misidentifying thelabels. But it may not be necessary to label every nucleotide type(i.e., adenine, cytosine, guanine, and thymine or uracil). For example,in one version three of four nucleotide types may be labeled while thefourth type remains unlabeled. During analysis of the electronmicroscope images, the three labeled nucleotide types may be located andthe fourth unlabeled nucleotide type may be identified by, for example,the absence of a label at locations where a nucleotide is expected basedon the locations of the labeled nucleotides.

Multiple strands of the same polymer may be labeled, a unique labelbeing applied to each of the different strands of the polymer. Eachlabel may identify a corresponding structural unit. Each of thesestrands may be imaged and evaluated separately. After the positions andidentities of the labels on each strand are evaluated, the label typeand position information from the multiple strands may be combined togenerate a sequence that contains all of the labeled structural units.For example, in the case of a nucleic acid, one strand may be labeled atits adenine (A) and cytosine (C) nucleotides, while the other strand islabeled at its guanine (G) and thymine (T) or uracil (U) nucleotides.The two strands may be imaged and evaluated separately. After thepositions and identities of the labels on each strand are evaluated, theinformation from the two strands may be combined into a full sequence.

FIG. 1 is a schematic diagram of an exemplary embodiment of a STEM 10that may be used for polymer sequencing. A specimen 20, which can beplaced in STEM 10 for imaging, may include a sample (not shown)containing a polymer to be imaged and a substrate (not shown) to supportthe sample across an area that can be exposed to an electron beam probe.The sample may include one or more polymer strands, such as strands ofDNA or RNA. However, the sample may be of any quantity, may be of anyshape or size, and may include any desired features. For example, thesample may include a specific configuration for a desired application orparameter setting. In another embodiment, the sample is a test sampleused for testing or optimization purposes, such as gold nano-particles.The substrate may include a layer of crystalline or amorphous carbon.Alternatively or in addition, the substrate may include boron nitride,silicon, silicon dioxide, aluminum, polymeric resins, or organicmaterials. Specimen 20 may be supported by a stage (not shown).

STEM 10 includes an electron beam source 30 to generate an electron beam40. Electron beam source 30 may be adapted to generate an electron beamhaving a current of less than about 100 mA. For example, for manyapplications electron beam source 30 may generate a beam current of fromabout 10 picoamps to about 1 milliamp. In an especially low-currentversion, however, electron beam source 30 may be adapted to generateelectron beam 40 to have a current of less than about 10 μA, such asless than about 10 pA.

Electron beam 40 travels from source 30 through an optical system. Theoptical system may define an optic axis 50 along which electron beam 40travels. The optical system may include illumination optics 60.Illumination optics 60 may include condenser lenses 70A-C to formelectron beam 40 into a collimated probe 80 that illuminates specimen20. Condenser lenses 70A-C may consist of, for example, two, three (asshown in the figure), or four lenses. Condenser lenses 70A-C may bemagnetic or electrostatic. Illumination optics 60 may also include anaberration corrector 90 to correct for aberrations of electron beam 40caused by the optical system.

The optical system of STEM 10 may also include an objective lens 100 tofocus electron beam 40. An objective aperture 110 may be provided in theback focal plane of objective lens 100 or a plane conjugate to the backfocal plane to define an acceptance angle, referring to an angle ofelectron beam 40 that is transmitted through aperture 110 and allowed toilluminate specimen 20. The rays that objective lens 100 focuses toprobe 80 on specimen 20 are thus limited in angle by aperture 110.

Larger acceptance angle may improve resolution. Because of thisrelationship between the acceptance angle and resolution of STEM 10, theacceptance angle can be selected based on the desired resolution. Forexample, if 1 Ångström resolution at 100 kilovolts is desired, it may bedesirable to have at least about 30 milliradians acceptance half-angle,or even at least about 40 milliradians acceptance half-angle. In oneexample, single-atom resolution—namely resolution at least as good asabout 0.3 nanometers and in some instances at least as good as about0.15 nanometers—may be desirable for a DNA-sequencing application.However, with an angular range that is unnecessarily high, current maybe wasted undesirably. Once a suitable accelerating voltage is chosen,the desired resolution may determine the acceptance angle of objectivelens 100.

Electron beam probe 80 may be conical or elongated along an axis. In oneversion, one or more of electron beam source 30 and condenser lenses70A-C are adapted to produce a longitudinally stretched electron beamprobe. For example, electron beam 40 may have a current distributionthat is approximately Gaussian along the longitudinal axis. In oneversion, the cross-section of electron beam 40 may be substantiallyelliptical.

FIG. 2 illustrates an example of an embodiment of an electron beam 40that is shaped into a longitudinally stretched probe 80A. In thisexample, the condenser lenses are multipoles 120A, 120B are combined tocondense a round beam into longitudinally stretched probe 80A. Electronbeam 40 is converged from a first cross-section 130A into a secondcross-section 130B that is substantially oblong and then, at specimen20, into longitudinally stretched probe 80A. At specimen 20,longitudinally stretched probe 80A is scanned along length of a sample140. FIG. 3A is a plot of an example of the current distribution of theelectron beam across the ‘A’ axis at the sample in FIG. 2, while FIG. 3Bis a plot of an example of the current distribution of the electron beamacross the ‘B’ axis.

FIGS. 4A and 4B illustrate another example of an electron beam 40 thatis shaped into a longitudinally stretched probe 80B. These figures showray diagrams for electron beam 40 from two orthogonal side views,respectively, in an optical system. The components shown are similar tothe optical components in FIG. 1, except that round condenser lenses70A-C are replaced with optical elements 150A-C, which may each containa combination of round lenses and multipoles. The flexibility of anillumination system created by breaking cylindrical symmetry allowsdifferent source demagnifications in multiple axes.

From the perspective of FIG. 4A along an ‘A’ axis, optical element 150Ais run with a stronger excitation to collect a relatively large range ofangles of electrons emitted from source 30 in objective aperture 90.Optical elements 150B-C form a relatively small beam that issubsequently sent into objective lens 100, forming a more parallel probe80B. Because of the larger range of angles collected from source 30, theelectrons that fill probe 80B are less coherent.

FIG. 4B illustrates a side view of the embodiment illustrated in FIG. 4Afrom a perspective along a ‘B’ axis that is orthogonal to the ‘A’ axis.From this perspective, probe 80B is similar to that created in FIG. 1using condenser lenses 70A-C to collect a small range of angles ofcoherent electrons emitted from source 30 in objective aperture 90, andsend them into objective lens 100 as a wide beam. Objective lens 100forms these rays into a high-resolution probe 80B in the ‘B’ axis, bythe inclusion of high-angle rays in the objective lens. The reducedcoherence and reduced ray angles included in the probe 80B causes it tobe larger in dimension along the ‘A’ axis than along the ‘B’ axis. Thisis demonstrated by the ray paths shown for electron beam 40 in FIGS. 4Aand 4B. Beam scanners 170, discussed in more detail below, are alsoshown in the figure.

The large amount of source demagnification along the ‘B’ axis, whencoupled with the small amount of source demagnification along the ‘A’axis, creates a filled electron beam with enhanced current that isformed into shaped probe 80B. In this case, relaxation of the resolutionalong one axis provides for increased current along that axis.

One or more beam scanners 170 may be provided to scan electron beam 40across specimen 20. Beam scanners 170 may scan electron beam 40 bygenerating either a magnetic or an electric field. For example, beamscanners 170 may include scan coils that generate an alternatingmagnetic field. Beam scanners 170 can be excited with ramp waveforms,causing the collimated probe to be scanned across the sample and therebyproducing an intensity signal at the detector unique to the location ofthe probe on the sample. FIG. 1 shows an example of electron beam 40being scanned between a first position 180A and a second position 180B.

Longitudinally stretched electron beam probe 80B can be used to scanpolymer strands of sample 140. For example, polymer strands may bescanned, one after the other, by longitudinally stretched probe 80B.FIG. 5 illustrates a schematic top view of an example of an embodimentof sample 140 that includes polymer strands on a substrate, and alongitudinally stretched electron beam probe scanning an area 160 thatcovers the length of one of the polymer strands. While probe 80B may bescanned along one axis, it may not be necessary, due to the shape ofprobe 80B, to scan the probe along an orthogonal axis in order to trackeach of the polymer strands. At each of the polymer strands, theextended length of stretched probe 80B may ensure that, all the wayalong the polymer strand, electron beam probe 80B illuminates thepolymer strand somewhere along its length. The polymer strand may besufficiently straightened on substrate 145 that its side-to-sidemovement along its length is smaller than the length of longitudinallystretched probe 80B. Sample 140 thus causes scattering of the electronbeam and its effect is present in the image. Accordingly, since scanningtransverse to the strand can be eliminated, the longitudinally stretchedshape can reduce the number of pixels needed for suitable imaging.

Returning to FIG. 1, the electron beam energy used in STEM 10 may bedetermined at least in part based on the transmission properties ofspecimen 20. The substrate may have a thickness on the order of 2nanometers, such as for example a thickness of about 1 nanometer. In oneexample, the substrate is made of carbon, although single-atomic-layergraphene may also be used. As a result, 1 keV electrons are likely to bethe lowest energy appropriate when considering voltage alone.

When specimen 20 is illuminated, electrons scatter from specimen 20,emerging from the other side in a pattern that is collected by one ormore detectors 190A, 190B (scattered rays not shown), 190C, 190D. Atomsof the sample having higher atomic number scatter the electrons tohigher angles, while lighter atoms scatter the electrons to lowerangles.

STEM 10 may have descanning and projection optics 200. The descanningoptics may de-scan scattered electron beams 210A, 210B, thus, forexample, realigning beam 210B with optic axis 50. The descanning opticsmay comprise, for example, descanning coils that may be symmetric toscan coils of beam scanners 170. The projection optics may includemagnifying lenses that allow additional manipulation of scatteredelectron beams 210A, 210B.

Detectors 190A-C are provided to detect electrons, such as electronbeams 210A, 210B, emerging from specimen 20 at one or more angles,respectively. Detectors 190A-C may be located on the side of specimen 20opposite from electron beam source 30. For example, detector 190A may beprovided to operate in a HAADF mode in which high-angle electron beam210A is detected, detector 190B may operate in a MADF mode, and detector190C may operate in a bright-field mode in which axial electron beam210B including a zero beam is detected. Detectors 190A-C may comprise,for example, a scintillator and a charge coupled device (CCD). Thescintillator may include one or more concentric annular detector ringsand a central circular disc detector in an approximately cylindricallysymmetric detector arrangement to receive the electrons. There may beapertures between detectors 190A-C. For each range of angles, detectors190A-C may provide an intensity signal corresponding to current receivedfor that angular range.

Alternate signals such as secondary electrons, backscattered electrons,and x-rays produced by the interaction between electron probe 80 and thesample may also be simultaneously detected in the region near the sampleby one or more detectors 190E.

The geometry of STEM 10 may be able to more efficiently collect coherentelectrons from source 30 compared to conventional TEM. Moreover, thegeometry of source 30 and detectors 190A, 190B may provide a relativelyhigh signal-to-noise ratio. The geometry of detector 190A distinguisheslow-angle scattering from high-angle scattering to make contrast in theimage depend on atomic number (Z). This efficiently acquires signal fromlabels that include heavy elements. This image data can be directlyinterpreted, unlike typical image data from conventional, phase-contrastTEM images, and may also have a higher signal-to-noise ratio. The use ofthe focused probe and annular detector geometry by STEM 10 alsointrinsically acts as a filter to increase image contrast between heavyand light elements of specimen 20. Thus, STEM 10 may be adapted to havea higher resolution than conventional, phase-contrast TEM (for example,about 1 Ångström), making it especially advantageous for single-atomlabels.

STEM 10 may be adapted to operate in a “bright field” mode in which adetector, such as detector 190C, detects a “forward-scattered” or“central” beam 210B of electrons emerging from specimen 20.Forward-scattered beam 210B refers to the zero beam (i.e., the 0scattering vector, referring to the beam whose direction is identical tothe orientation of beam 40 impinging on specimen 20) and a small rangeof angles around the zero beam. The bright-field mode may beparticularly sensitive to the energy loss of the electrons, indicatingchemical composition. These electrons can be detected to determine, forexample, bonding energies of molecules that compose the sample.

In one version, a detector 190D may be provided to detect electrons inone or more preselected range of energies. Coupling optics 220 may beprovided and detector 190D may include an electron prism 230 to filterout electrons that are not in the preselected energy ranges. In oneversion, this is used for electron energy loss spectroscopy (EELS).Electron prism 230 may, for example, generate an electric or magneticfield by using electrostatic or magnetic means, respectively. The fieldstrength and dimensions of electron prism 230 may be selected such that,when the electrons of varying energies pass through the field, theelectrons in the preselected energy range are transmitted throughelectron prism 230 while the remaining electrons are blocked. Detector190D may also include a receiver 240, such as including a scintillatorand CCD, to receive the transmitted electrons and convert that currentinto a detection signal. The EELS detection signal can be expressed as aplot 250 of current as a function of electron energy loss.

Alternatively to the bright-field mode, the STEM may be adapted tooperate in a dark-field mode in which one or more electron beams 210Aemerging from specimen 20 within a particular angular range aredetected. Since specimen 20 is illuminated at approximately a point,this angular range of detection can be tightly controlled. For example,the dark-field mode may be an annular-dark-field (ADF) mode in which anelectron beam shaped as a hollow cone of preselected thickness isdetected. The dark-field mode may involve detecting a hollow cone athigher angles, which is referred to as high-angle annular-dark-field(HAADF) mode. The dark-field mode may also be a medium-angle dark-field(MADF) mode, in which a range of angles between the bright-field modeand the HAADF mode are detected. These dark-field modes can produce animage with monotonic contrast change with increasing atomic number,which enables direct interpretability of the image to determine relativeatomic weights. For example, dark-field imaging can be used to obtainchemically sensitive projections of single atoms, clusters of atoms, ornanostructures. STEM 10 can also operate in simultaneous bright-fieldand dark-field modes. An electron beam source having a high-brightnessgun may allow this mode to operate faster.

Each of detectors 190A, 190B in a cylindrically symmetric arrangementmay limit the scattered electrons to an angular range denoted here asφ_(d), which defines an annulus between an inner angle φ₁ and outerangle φ₂. For an ADF mode these angles may be, for example, from about25 mrad to about 60 mrad for φ₁, and from about 60 mrad to about 80 mradfor φ₂. For HAADF mode using detector 190A, these angles may be, forexample, from about 60 mrad to about 80 mrad for φ₁, and greater thanabout 100 mrad for φ₂.

Alternatively to a cylindrically symmetric system, one or more ofdetectors 190A, 190B may have a shape that is cylindrically asymmetric.For example, detectors 190A, 190B may have an inner or outer perimeterthat is polygonal, such as square or hexagonal. Alternatively, thesedetectors may have another shape that is cylindrically asymmetric.

In one version, the stage is moved continuously while electron beam 40is simultaneously scanned. This may improve throughput by allowingcontinuous acquisition of images while eliminating the settling timecaused by stop-start motion of a stage that is moved discretely and thatmay prevent acquisition of a still image of the sample. For example, apiezoelectric stage may be used. The piezoelectric stage may be able tomove very quickly and smoothly so that short exposures on the order ofmilliseconds or microseconds can be practically achieved. Thepiezoelectric stage may also be adapted to move the stage with very highpositional precision. Furthermore, the throughput of data that emergesfrom the detectors may be substantial, such that electronics capable ofdealing with this data throughput downstream of the detectors may bedesirable. In one embodiment, the stage motor is capable of displacingthe specimen at a speed of at least about 100 nm per second.

In order to improve speed, accuracy, and sensitivity, aberrationcorrector 90 may correct for aberrations in electron beam 40, such asspherical aberrations and parasitic aberrations. The parasiticaberrations may or may not be cylindrically symmetric. Aberrationcorrector 90 may include electromagnetic lenses to correct for theseaberrations. Parasitic aberrations may be caused, for example, by theoptical elements having been machined in such a way as to be veryslightly off-axis or very slightly non-round. Examples of commerciallyavailable aberration correctors for a STEM include Nion Co.quadrupole-octupole correctors (available from Nion Company of Kirkland,Wash.) and CEOS sextupole or quadrupole-octupole correctors (availablefrom Corrected Electron Optical Systems GmbH of Heidelberg, Germany). Inone example, aberration correction may improve resolution from about 2to 3 Ångströms to about 1 Ångström. Labels that include clusters ofatoms may be suitably imaged at 2 to 3 Ångströms. For labels thatconsist of single atoms, 1 Ångström resolution may be preferable.

STEM 10 may include a controller (not shown) to control operation ofSTEM 10. The controller may, for example, receive inputs from a humanuser, provide instructions to STEM 10, and/or perform data processing ofimages generated by STEM 10. The controller may automatically controlone or more aspects of operation of STEM 10, and may even be adapted toentirely automate the operation of STEM 10. The controller may controlthe components of the optical system, such as beam scanners 170 and thestage. Alternatively or in addition, the controller may receive one ormore images from detectors 190A-D to be processed computationally. Forexample, the controller may process collected particle data and/orprocess any desired images. The controller may include an imageformation unit for this purpose. The image formation unit may bedisposed within or external to the STEM column and communicate with theoptical system and the stage in any fashion, such as by a direct orindirect electronic coupling, or via a network.

The controller may include one or more microprocessors, controllers,processing systems, and/or circuitry, such as any combination ofhardware and/or software modules. For example, the controller may beimplemented in any quantity of personal computers, such asIBM-compatible, Apple, Macintosh, Android, or other computer platforms.The controller may also include any commercially available operatingsystem software, such as Windows, OS/2, Unix, or Linux, or anycommercially available and/or custom software such as communicationssoftware or microscope monitoring software. Furthermore, the controllermay include one or more types of input devices, such as for example atouchpad, keyboard, mouse, microphone, or voice recognition.

The controller software, such as a monitoring module, may be stored on acomputer-readable medium, such as a magnetic, optical, magneto-optic, orflash medium, floppy diskettes, CD-ROM, DVD, or other memory devices,for use on stand-alone systems or systems connected by a network orother communications medium, and/or may be downloaded, such as in theform of carrier waves, or packets, to systems via a network or othercommunications medium.

The controller can be adapted to automatically diagnose the magnitudesof various aberrations and apply compensating signals to the opticalsystem, such as to aberration-producing lens elements and aberrationcorrector 90. One exemplary method is to raster scan one or more tuningregions of specimen 20 to generate an image and to analyze the generatedimage to extract information about aberrations that can be used tocorrect the aberrations. The tuning regions may be of any shape or sizeand may be located within or without the areas to be scanned. Anotherexemplary method is to acquire one or more images as a function ofillumination tilt and defocus, and to extract the blurring effect of thetilt and defocus. The blurring gives a value for the defocus andastigmatism at a variety of angles. This process can provide sufficientdata to numerically compute an aberration function for the imagingsystem. Yet another method is to defocus electron beam 40 and usebright-field detector 190C, such as a CCD camera, to generate aRonchigram image, or a plurality of Ronchigram images taken at differentpositions of specimen 20, and then refocus electron beam 40 forcontinued imaging of the sample. The Ronchigram image can providesufficient aberration information to derive optical parameters thatpermit suitable compensation for these aberrations.

A sample used for the purposes of diagnosing aberrations may containsingle atoms or clusters of atoms, or may be another kind of sample madefor the purpose of diagnosing aberrations. For example, the sample maybe specimen 20 that is also the subject of interest for study.Alternatively, the sample may be a sample used solely for calibration ofSTEM 10.

STEM 10 may include or be connected to a power supply that providespower to components of STEM 10, such as electron beam source 30,condenser lenses 70A-C, objective lens 100, detectors 190A, 190B, thestage, and the controller. In one embodiment, the optical system of STEM10 has a total power consumption of less than about 2.5 kW.

The speed and quality of STEM imaging can be improved to make itadvantageous for polymer sequencing. First, STEM 10 may use alongitudinally stretched probe, such as described above. Second, STEM 10may selectively direct the imaging along the sample. In this way, speedcan be improved by from about 10 to about 100,000 times when compared toconventional TEM or STEM imaging. Third, STEM 10 may automatically tunethe optical system one or more times during imaging, such as to refocusand compensate for aberrations.

STEM 10 may have a characteristic area at the plane of specimen 20 inwhich optical characteristics, such as, for example, resolution, areselected to be within a range suited to the imaging that is performed.This area may be referred to as the “field of view” of STEM 10. In oneembodiment, for example, the field of view is substantially free of“coma,” a second-order type of parasitic aberration, and/or astigmatism.In one example provided solely for the purpose of illustration, thefield of view has a diameter of about 1 to about 2 microns. Within thefield of view, electron beam 40 may be scanned in one or more scanningareas across specimen 20 by electronic shifting, such as by generatingan electric or magnetic field, while remaining within the desired rangeof optical characteristics (such as high resolution).

FIG. 6 illustrates an example of an embodiment in which electron beam 40is electronically shifted from a first position 180A to a secondposition 180B on specimen 20, such as to scan electron beam 40 from afirst location to a second location to move electron beam 40 between twodifferent samples. As shown in the figure, beam scanners 170 maygenerate a magnetic or electric field that deflects electron beam 40. Ifan area of specimen 20 that is currently outside the field of view is tobe scanned, that area can be brought into the field of view, such as bymoving the stage carrying specimen 20. FIG. 7 illustrates an example ofan embodiment of a stage 270 supporting specimen 20. In this example,stage 270 can be moved back and forth along two orthogonal axes inrelation to the rest of the microscope by one or more stage motors (notshown). To scan one or more regions that fall outside the dimensions ofa single field of view, stage 270 may be moved either between imagingcycles or simultaneous with continuous imaging. Stage 270 may also bemoved along a third axis that is orthogonal to the other two axes, suchas a “height” axis substantially parallel to the optic axis. Defocus maybe accomplished by adjusting the stage position on this third axis,which may be done rather than adjusting optical parameters, for examplesignals applied to the condenser and objective lenses. Furthermore, thebeam scanners and stage motors may be used together, such as where thestage motors are used to move the stage continuously during imagingalong one axis while the beam scanners raster scan electron beam 40along an approximately orthogonal axis.

Within each field of view, the STEM may define one or more scanningareas in which the electron beam will be scanned to contribute to thefinal image. The STEM may perform the imaging of sample 140 in one ormore cycles corresponding to the scanning areas, each imaging cycle fora scanning area yielding a contribution that is referred to here as asub-image. Each scanning area may be noncontiguous, contiguous, oroverlapping in relation to scanning areas within the same field of viewor scanning areas in different fields of view. Moreover, the scanningareas may even be a combination of noncontiguous (i.e., with edgesseparated by a space), contiguous (i.e., edge to edge), or overlapping.

In one example provided solely for the purpose of illustration, themicroscope has a resolution of about 1 Ångström. For a DNA strand, inone embodiment in which a heavy label is attached to a single type ofnucleotide, one may expect the presence of a heavy label at about every15 to about every 25 Ångströms. Multiple scanning areas may be definedwithin the field of view, each scanning area having dimensions of fromabout 500 pixels by about 500 pixels to about 2000 pixels by about 2000pixels. For example, each scanning area may have rectangular dimensionsof about 500 pixels by about 2000 pixels. In another example, eachscanning area is a strip with dimensions of about 5 pixels by about 8000pixels.

Noncontiguity between certain scanning areas, such as resulting fromdefining the scanning areas to track different strands of sample 140,may yield an increase in speed by allowing the imaging process to avoidunnecessary scanning, such as unnecessary scanning of “empty” regionswhere portions of sample 140 are not actually present. For example, thecontroller may define scanning areas that track, in two dimensions inthe plane of specimen 20, the location of a sample that consists ofmultiple DNA strands that have been straightened out across substrate145. In one embodiment, noncontiguity of scanning areas may allow theSTEM to operate at least 100 times faster than a conventional STEM.

Overlapping scanning areas, meanwhile, may provide imaging redundancythat can be used to ensure accuracy of the eventual sequencing results.In one embodiment, overlapping of scanning areas corresponding todifferent stage positions may be desirable. This may be advantageous,for example, if hysteresis in the movement of stage 270 is present suchthat stage positioning is inexact in relation to the resolution ofimaging. In this case, redundancy in imaging may make up for theinexactitude of positioning. For scanning areas within the same field ofview, between which the electron beam is electronically shifted, beampositioning may be sufficiently exact to use scanning areas that arecontiguous or overlap by a smaller amount.

The STEM may perform a preliminary imaging of sample 140 before the mainimaging of sample 140 that is used to obtain the sequence information.The preliminary imaging may be, for example, a faster, lower-resolution,or lower-dose scan of sample 140 used to determine the location ofsample 140, such as the locations of each of multiple polymer strandsthat make up sample 140. This scan may, for example, cover asubstantially contiguous area, rather than being limited to particular,discrete scanning areas. Surveying may also be performed outside of theSTEM, where fiducials on specimen 20 or another registration mechanismis provided, such as using a scanning electron microscope (SEM) oralternatively an optical microscope (such as for fluorescent orlight-visible markers). The controller may then define the scanningareas such that the scanning areas track sample 140 based on thedetermined location of sample 140. Using the scanning areas, thecontroller may perform a slower or higher-resolution scan within thescanning areas, thus concentrating the imaging on the actual location ofsample 140 and thereby improving efficiency. For example, within eachscanning area the microscope may raster scan the electron beam.

After the sub-images are obtained, the controller may stitch togetherthe sub-images to produce a partially or wholly comprehensive image ofsample 140. For example, where there are overlapping or contiguoussub-images, these sub-images may be joined together to yield imagingdata that is continuous across the corresponding scanning areas. Foroverlapping sub-images, the controller may use the redundant imageinformation at the overlap to accurately join the sub-images togetherinto a comprehensive image. In this way, the sequence information fromthe polymer can be obtained without gaps or inaccurate repetitions.

Tracking the sample may become even more advantageous when the polymerstrands of sample 140 are less straightened. A polymer strand with morecurvature and that does not overlap with itself, for example, may occupymore area on the substrate in relation to its length. The STEM, bydefining scanning areas that track the paths of the polymer strands, maybe able to avoid even more empty area where the polymer strands are notpresent, providing faster imaging speeds.

The controller may evaluate information originating at one or more ofthe detectors, either between imaging cycles or simultaneous withimaging, to determine the current quality of imaging. In one version,imaging information from dedicated “tuning regions” is used. However,the images themselves may additionally or alternatively be used. Forexample, information from the most recent images can be used todetermine trends of tuning deterioration. This evaluation can be used toset optical parameters of the STEM to improve the quality of imaging.For example, returning to FIG. 1, the optical parameters may be appliedto condenser lenses 70A-C, objective lens 100, aberration corrector 90,and the stage. For example, the optical parameters applied to condenserlenses 70A-C, objective lens 100, and the stage may improve the focus,while the optical parameters used on aberration corrector 90 maycompensate for higher orders of aberration. This process may be referredto as “re-tuning” the microscope.

It may be desirable to maintain the microscope in a substantially steadystate in terms of contamination and stability during imaging. But theperformance of the optical system may tend to deteriorate over time. Inone example, the optical system may deteriorate to an undesirable statein from about 5 to about 30 minutes, such as about 15 minutes. When thishappens, it may become advantageous to perform re-tuning. In oneversion, first-order and second-order aberrations may be especiallyprone to deterioration and/or advantageous to compensate for byre-tuning. The electron beam source may also deteriorate over time. Torefresh the electron beam source, it can be “flashed” by running a highcurrent through it between beam scanning cycles. This causes a localizedheating of the filament that reconditions the source.

Re-tuning may be triggered according to any suitable procedure. Thecontroller may monitor the microscope to initiate the determination ofimaging quality, the controller may automatically initiate re-tuning atregular intervals, or the controller may poll a store of recentlygenerated images to determine image quality as a background process. Forexample, the re-tuning may be triggered within any desired timeinterval, such as within any quantity of hours or minutes, or subsequentto any quantity of images generated by the microscope or every Nthlinear scan or scan cycle performed by the microscope. In an exemplaryembodiment, the controller initiates re-tuning between sequential fieldsof view. In another embodiment, however, the controller can re-tune theoptical system between sequential scanning areas.

At each of the sub-areas, STEM 10 may scan and image a tuning regionwithin or without the sub-area one or more times to generate one or moresub-images that can be used to track the sample and/or produce imagingmetadata. The imaging metadata may include, for example, focus error andamounts of various orders of aberration, and beam current. Thecontroller may use the imaging metadata to generate optical parametersto improve image quality, such as, for example, to autofocus the imageat the elevation of sub-area. For example, the controller may evaluateseveral sub-images taken in a particular area to determine the magnitudeand direction of focus error. Using this information, STEM 10 cangenerate a final well-focused sub-image that will be used for evaluationof the sample itself. STEM 10 may use any number of sub-images of asample to determine imaging metadata. The sub-images may cover anydesired variation range for a particular parameter.

In analyzing an image, the controller may analyze any suitablecharacteristics of the image, such as intensity, pixel counts, or power,each of which may be analyzed in real space or in frequency space (sothat intensity may be within or without a spatial frequency range). Whencomparing images or evaluating a series of images, the controllerutilizes any characteristic that differs between the images, such as ina preselected region of the images.

The controller may also use any number of images for the image qualitycomparison, where the image quality values for current and prior imagesmay be combined in any suitable fashion, such as averaged, weighted, orsummed. A user threshold for image quality may be set to any suitablevalue. A comparison of image quality values may utilize any mathematicalor statistical operations to determine image quality compliance, such asa comparison, statistical variance, or deviation.

The STEM imaging process may be performed entirely automatically, suchas after initiation by a user or initiation by a larger process of whichthe STEM imaging is a subprocess. Parameters may be determinedautomatically and applied to the microscope. Alternatively, any part ofthe technique, such as scanning of images, determination of parameters,or application of the parameters, may be performed manually. Forexample, the computer system may provide the optimal settings to atechnician that manually applies the settings to the microscope. Themicroscope controller may perform any desired processing, such asmonitoring and adjustment of optical parameters or image formation andprocessing. For example, the controller may align images using imageregistration algorithms. The controller may also adjust the aberrationsand defocus of an image based on characteristics of a previous image.

In one exemplary embodiment, imaging of the scanning areas may proceedas follows. The controller may initiate imaging by STEM 10 by scanning afirst scanning area in a first field of view. For example, the electronbeam may be raster scanned throughout the first scanning area. Moreover,the scanning area may be scanned more than once, such as to trydifferent optical parameters or otherwise improve imaging quality. Whena useful sub-image for the first scanning area has been generated, thecontroller may control the microscope to begin scanning a secondscanning area in the same field of view, if there is any. After all thescanning areas in the field of view have been imaged, the stage may bemoved into a new field of view. The process described above may berepeated until all planned fields of view have been scanned.

Alternatively to sequentially scanning the scanning areas that are inthe same field of view, moving from one scanning area to the next afterscanning is complete for the previous scanning area, the controller maycontrol STEM 10 to scan multiple scanning areas in parallel, such thatmore than one scanning area is scanned before any of them has beencompletely scanned. In one exemplary embodiment, STEM 10 may scanproximate regions of multiple scanning areas before completing thescanning of any of these scanning areas. This may be advantageous, forexample, where the scanning areas are shaped as elongated strips linedup in parallel, each elongated strip corresponding to a polymer strand,such that the strips are of different length as constrained by theboundary of the field of view.

The set of sub-images that coincide with the length of the sample may bestitched together into a comprehensive image. Automatic re-tuning may beable to yield, without substantial user intervention, a high-qualitycomprehensive image suitable for extracting the sequence information ofthe polymer. The controller can then analyze the image to identify thestructural units of the polymer being sequenced, such as by identifyingthe labels attached to the polymer.

FIG. 8 illustrates an example of an embodiment of a field of view 280enclosing a relatively large scanning area 160, such as nearly the sizeof field of view 280, that covers a region of sample 80 that includesmultiple polymer strands. A tuning region 290A inside scanning area 160may initially be scanned to obtain optical parameters useful forautomatically re-tuning the optical system of the microscope.Alternatively, a tuning region 290B outside the scanning area 160 may berelied on. However, it may be desirable to locate tuning region 290A,290B to avoid covering the polymer strands themselves, such that thestrands are not damaged by electron beam illumination. In oneembodiment, the large size of scanning area 160 shown in this figure maybe used at reduced resolution for a low-dose, high-speed survey tolocate strands of sample 80. However, scanning area 160 does not have tobe nearly the full size of field of view 280. It may be advantageous forscanning area 160 not to be of maximal size, since tuning drift mayotherwise vignette the corners of scanning area 160. For example,scanning area 160 may have a size that is about 10 to about 20 timessmaller in diameter than field of view 280.

FIG. 9 illustrates another perspective on scanning area 160 of FIG. 8,showing a plurality of scanning areas 160 that overlap at overlap areas300. As shown in the figure, scanning areas 160 are arranged to trackthe sample, thereby improving imaging efficiency. Imaging informationobtained redundantly from overlap areas 300 can be used to stitchtogether sub-images obtained from the different scanning areas 160, andto perform drift compensation and drift distortion correction. Theelectron beam may be moved between scanning areas 160 either by movingthe stage or by electronically shifting the electron beam.

FIG. 10 illustrates another example of an embodiment of a plurality offields of view 280. In this example, multiple scanning areas 160 aredefined within each field of view 280. Scanning areas 160 may be shapedas elongated strips that overlie the polymer strands of the sample.Since scanning is confined to scanning areas 160, rather than having toscan the entire field of view 280, including the spaces between scanningareas 160, imaging speed is improved. Within each field of view 280,scanning areas 160 may be scanned sequentially in any order, or even inparallel (i.e., scanning parts of different scanning areas 160 beforeone of scanning areas has been completely scanned), as suited to theapplication. For example, the scanning order may be adapted to furtherimprove the speed of imaging. The stage may be displaced to move along apath 310, from one field of view to the next, such as shown in thefigure. Path 310 may be selected to provide suitably good coverage ofthe sample in a substantially efficient way. Within each of fields ofview 280, the electron beam may be electronically shifted to scan acrossscanning areas 160.

FIG. 11 illustrates yet another example of an embodiment of a field ofview 280 enclosing a plurality of scanning areas 160. In this example,scanning areas 160 are arranged in elongated strips that overlie thepolymer strands of sample 140. However, unlike the example illustratedin FIG. 10, in this figure a plurality of contiguous scanning areas formeach of the elongated strips. Between the elongated strips, scanningareas 160 are noncontiguous, separated by spaces that do not have to bescanned, thereby improving imaging speed. Within field of view 280,scanning areas 160 may be scanned sequentially in any order, or even inparallel, as suited to the application.

FIG. 12 illustrates an example in which information 320 about thepresence or absence of labels corresponding to particular structuralunits at particular positions along the lengths of a number of strandsof a polymer are converted into the sequence of the polymer. In the casewhere the polymer is a nucleic acid, such as in the example illustrated,the labels correspond to particular nucleotide types (A, G, T, C). Inthe figure, each of the rows shows two portions of label informationfrom imaged strands. The portions of label information may come fromseparate strands of the nucleic acid or different noncontiguous scanningareas. Using bioinformatics techniques, these portions of labelinformation can be arranged as a function of corresponding position inthe nucleic acid and then connected together. By combining thisnucleotide information, sequence 330 of the nucleic acid can bedetermined. Where sub-images from two scanning areas have previouslybeen graphically stitched together, as described above, there may not beany need to rely on bioinformatics techniques to connect the labelinformation from the sub-images since the label information may alreadybe continuous.

The STEM may be used in any suitable facility in any desiredarrangement, such as networked, direct, or indirect communicationarrangements. Moreover, the various functions of the STEM may bedistributed in any manner among any quantity of components, such as oneor more hardware and/or software modules or units. The hardware mayinclude microscopes, machine managers, computer or processing systems,circuitry, networks, and image stores, that may be disposed locally orremotely of each other and may communicate with each other or be coupledto each other in any suitable manner, such as wired or wireless, over anetwork such as WAN, LAN, Intranet, Internet, hardwire, or modem,directly or indirectly, locally or remotely from each other, via anycommunications medium, and utilizing any suitable communication protocolor standard. The software and/or algorithms described above may bemodified in any manner that accomplishes the functions described herein.

The embodiments of the STEM described herein may be implemented witheither electrostatic or magnetic components. The STEM may include anyquantity of electrostatic or magnetic components, such as an electron orother particle gun, lenses, a dispersion device, stigmator coils,electron detectors, and stages, arranged within or without the STEM inany suitable fashion. Image stores, files, and folders used by the STEMsystem may be of any quantity and may be implemented by any storagedevices, such as memory, database, or data structures.

Implementation of aspects of the STEM, such as the image processing oraberration correction, may be distributed among the controller or otherprocessing devices in any desired manner, where these devices may belocal or remote in relation to one another. The controller maycommunicate with and/or control the microscope to perform any desiredfunctions, such as scanning the specimen and generating the images ortransferring images to memory.

Although the foregoing embodiments have been described in detail by wayof illustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art in lightof the description herein that certain changes and modifications may bemade thereto without departing from the spirit or scope of the appendedclaims. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only,” and the like in connection with therecitation of claim elements, or use of a “negative” limitation. As willbe apparent to those of ordinary skill in the art upon reading thisdisclosure, each of the individual aspects described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalaspects without departing from the scope or spirit of the disclosure.Any recited method can be carried out in the order of events recited orin any other order which is logically possible. Accordingly, thepreceding merely provides illustrative examples. It will be appreciatedthat those of ordinary skill in the art will be able to devise variousarrangements which, although not explicitly described or shown herein,embody the principles of the disclosure and are included within itsspirit and scope.

Furthermore, all examples and conditional language recited herein areprincipally intended to aid the reader in understanding the principlesof the invention and the concepts contributed by the inventors tofurthering the art, and are to be construed without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles and aspects of the invention, as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryconfigurations shown and described herein. Rather, the scope and spiritof present invention is embodied by the claims.

In this specification, various preferred embodiments have been describedwith reference to the accompanying drawings. It will be apparent,however, that various other modifications and changes may be madethereto and additional embodiments may be implemented without departingfrom the broader scope of the claims that follow. The specification anddrawings are accordingly to be regarded in an illustrative rather thanrestrictive sense.

We claim:
 1. A scanning transmission electron microscope for imaging aspecimen, the microscope comprising: an electron beam source to generatean electron beam; beam optics to converge the electron beam; a stage tohold a specimen in the path of the electron beam; a beam scanner to scanthe electron beam across the specimen; a detector to detect electronstransmitted through the specimen to generate an image; and a controllerto (1) define one or more scanning areas corresponding to locations ofthe specimen, (2) control one or more of the beam scanner and stage toselectively scan the electron beam in the scanning areas, (3) generate asub-image for each of the scanning areas, (4) stitch together thesub-images for the scanning areas to generate a stitched-together image,and (5) analyze the stitched-together image to determine informationregarding the specimen.
 2. The scanning transmission electron microscopeof claim 1, wherein the controller is adapted to perform one or more of(i) drift compensation and (ii) drift distortion correction to generatethe stitched-together image.
 3. The scanning transmission electronmicroscope of claim 1, wherein the plurality of the scanning areasoverlap, and wherein the controller is adapted to generate the sub-imagefor each of the scanning areas and stitch together the sub-images into acomprehensive image based on matching imaging information from the areaof overlap.
 4. The scanning transmission electron microscope of claim 1,wherein the plurality of the scanning areas are contiguous, and whereinthe controller is adapted to generate the sub-image for each of thescanning areas and stitch together the sub-images into a comprehensiveimage based on matching imaging information from the areas near thecontiguity.
 5. The scanning transmission electron microscope of claim 1,wherein the specimen comprises an elongated object, and wherein thecontroller is adapted to control the microscope to selectively scan theelectron beam substantially along the elongated object of the specimen.6. The scanning transmission electron microscope of claim 1, comprisingone or more detectors to detect electrons transmitted through thespecimen to generate the image, and wherein the detectors and controllerare adapted to tune the beam optics during imaging.
 7. The scanningtransmission electron microscope of claim 6, wherein the detectors andcontroller are adapted to tune the beam optics between cycles ofscanning the electron beam.
 8. The scanning transmission electronmicroscope of claim 6, wherein the detectors and controller are adaptedto tune the beam optics between cycles of scanning areas correspondingto sub-images and generating the sub-images.
 9. The scanningtransmission electron microscope of claim 1, wherein the beam optics areadapted to converge the electron beam into a longitudinally stretchedbeam.
 10. The scanning transmission electron microscope of claim 1,wherein the controller is adapted to define one or more of the scanningareas to be displaced in at least two dimensions in relation to eachother, and wherein the controller is adapted to stitch together thesub-images by feature registration in at least two dimensions.
 11. Amethod of imaging a specimen, the method comprising: generating anelectron beam; converging the electron beam; holding a specimen on astage in the path of the electron beam; defining one or more scanningareas corresponding to locations of the specimen; controlling one ormore of the beam scanner and the stage to selectively scan the electronbeam in the scanning areas; detecting electrons transmitted through thespecimen to generate a sub-image for each of the scanning areas;stitching together the sub-images for the scanning areas to generate astitched-together image; and analyzing the stitched-together image todetermine information regarding the specimen.
 12. The method of claim11, comprising performing one or more of (i) drift compensation and (ii)drift distortion correction to generate the stitched-together image. 13.The method of claim 11, wherein the plurality of the scanning areasoverlap, and comprising generating the sub-image for each of thescanning areas and stitching together the sub-images into acomprehensive image based on matching imaging information from the areaof overlap.
 14. The method of claim 11, wherein the plurality of thescanning areas are contiguous, and comprising generating the sub-imagefor each of the scanning areas and stitching together the sub-imagesinto a comprehensive image based on matching imaging information fromthe areas near the contiguity.
 15. The method of claim 11, comprisingdefining one or more of the scanning areas to be displaced in at leasttwo dimensions in relation to each other, and comprising stitchingtogether the sub-images by feature registration in at least twodimensions.
 16. The method of claim 11, further comprising tuning thebeam optics between cycles of scanning areas corresponding to sub-imagesand generating the sub-images.
 17. The method of claim 11, comprisingcontrolling the microscope to selectively scan the electron beam in aplurality of scanning areas of the specimen where one or more proteinsare located, and comprising analyzing the image to determine informationregarding the proteins.
 18. The method of claim 11, wherein convergingthe electron beam comprises converging the electron beam into alongitudinally stretched beam.
 19. A scanning transmission electronmicroscope for imaging a specimen, the microscope comprising: anelectron beam source to generate an electron beam; beam optics toconverge the electron beam into a longitudinally stretched beam; a stageto hold a specimen in the path of the electron beam; a beam scanner toscan the electron beam across the specimen; a detector to detectelectrons transmitted through the specimen to generate an image; and acontroller to (1) define one or more scanning areas corresponding tolocations of the specimen, (2) control one or more of the beam scannerand stage to selectively scan the electron beam in the scanning areas,(3) generate a sub-image for each of the scanning areas, (4) stitchtogether the sub-images for the scanning areas to generate astitched-together image, and (5) analyze the stitched-together image todetermine information regarding the specimen.
 20. The scanningtransmission electron microscope of claim 19, wherein the controller isadapted to perform one or more of (i) drift compensation and (ii) driftdistortion correction to generate the stitched-together image.